Accumulation of environmental chitin in the lungs can lead to pulmonary fibrosis, characterized by inflammatory infiltration and fibrosis in acidic chitinase (Chia)-deficient mice. Transgenic expression of Chia in these mice ameliorated the symptoms, indicating the potential of enzyme supplementation as a promising therapeutic strategy for related lung diseases. This study focuses on utilizing hyperactivated human Chia, which exhibits low activity. We achieved significant activation of human Chia by incorporating nine amino acids derived from the crab-eating monkey (Macaca fascicularis) Chia, known for its robust chitin-degrading activity. The modified human Chia retained high activity across a broad pH spectrum and exhibited enhanced thermal stability. The amino acid substitutions associated with hyperactivation of human Chia activity occurred species specifically in monkey Chia. This discovery highlights the potential of hyperactivated Chia in treating pulmonary diseases resulting from chitin accumulation in human lungs.

• Keywords
• acidic chitinase (Chia), amino acid substitutions, chitin, enzyme engineering, evolution, exon swapping, hyperactivation, primate lineage, treating pulmonary diseases,
• MeSH
• Chitin metabolism   MeSH
• Chitinases * genetics   metabolism   chemistry   MeSH
• Hydrogen-Ion Concentration MeSH
• Humans MeSH
• Mice MeSH
• Enzyme Stability MeSH
• Amino Acid Substitution MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CHIA protein, human MeSH Browser
• Chitin MeSH
• Chitinases * MeSH

YKL-40, also known as human cartilage glycoprotein-39 (HC-gp39) or CHI3L1, shares structural similarities with chitotriosidase (CHIT1), an active chitinase, but lacks chitinase activity. Despite being a biomarker for inflammatory disorders and cancer, the reasons for YKL-40's inert chitinase function have remained elusive. This study reveals that the loss of chitinase activity in YKL-40 has risen from multiple sequence modifications influencing its chitin affinity. Contrary to the common belief associating the lack of chitinase activity with amino acid substitutions in the catalytic motif, attempts to activate YKL-40 by creating two amino acid mutations in the catalytic motif (MT-YKL-40) proved ineffective. Subsequent exploration that included creating chimeras of MT-YKL-40 and CHIT1 catalytic domains (CatDs) identified key exons responsible for YKL-40 inactivation. Introducing YKL-40 exons 3, 6, or 8 into CHIT1 CatD resulted in chitinase inactivation. Conversely, incorporating CHIT1 exons 3, 6, and 8 into MT-YKL-40 led to its activation. Our recombinant proteins exhibited properly formed disulfide bonds, affirming a defined structure in active molecules. Biochemical and evolutionary analysis indicated that the reduced chitinase activity of MT-YKL-40 correlates with specific amino acids in exon 3. M61I and T69W substitutions in CHIT1 CatD diminished chitinase activity and increased chitin binding. Conversely, substituting I61 with M and W69 with T in MT-YKL-40 triggered chitinase activity while reducing the chitin-binding activity. Thus, W69 plays a crucial role in a unique subsite within YKL-40. These findings emphasize that YKL-40, though retaining the structural framework of a mammalian chitinase, has evolved to recognize chitin while surrendering chitinase activity.

• Keywords
• YKL-40, asthma, biomarker, cartilage biology, chitin, chitin-binding, chitinase, enzyme inactivation, inflammation, tumor marker,
• MeSH
• Chitin * metabolism   chemistry   MeSH
• Chitinases metabolism   genetics   chemistry   MeSH
• Exons MeSH
• Hexosaminidases metabolism   chemistry   genetics   MeSH
• Catalytic Domain MeSH
• Humans MeSH
• Evolution, Molecular MeSH
• Chitinase-3-Like Protein 1 * metabolism   genetics   chemistry   MeSH
• Amino Acid Sequence MeSH
• Amino Acid Substitution MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CHI3L1 protein, human MeSH Browser
• Chitin * MeSH
• Chitinases MeSH
• chitotriosidase MeSH Browser
• Hexosaminidases MeSH
• Chitinase-3-Like Protein 1 * MeSH